title: Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax.
abstract: The 28S rRNA gene was amplified and sequenced from P. falciparum and P. vivax isolates collected from northwest India. Based upon the sequence diversity of the Plasmodium 28SrRNA gene in comparison with its human counterpart, various nested polymerase chain reaction (PCR) primers were designed from the 3R region of the 28SrRNA gene and evaluated on field isolates. This is the first report demonstrating the utility of this gene for species-specific diagnosis of malaria for these two species, prevalent in India. The initial evaluation on 363 clinical isolates indicated that, in comparison with microscopy, which showed sensitivity and specificity of 85·39% and 100% respectively, the sensitivity and specificity of the nested PCR assay was found to be 99·08% and 100% respectively. This assay was also successful in detecting mixed infections that are undetected by microscopy. Our results demonstrate the utility of the 28S rRNA gene as a diagnostic target for the detection of the major plasmodial species infecting humans.
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science, Pilani, Rajasthan, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Pakalapati
authors.forename: Shilpi
authors.initials: S
authors.lastname: Garg
authors.forename: Sheetal
authors.initials: S
authors.lastname: Middha
authors.forename: Jyoti
authors.initials: J
authors.lastname: Acharya
authors.forename: Amit K
authors.initials: AK
authors.lastname: Subudhi
authors.forename: Arunachalam P
authors.initials: AP
authors.lastname: Boopathi
authors.forename: Vishal
authors.initials: V
authors.lastname: Saxena
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Kochar
authors.forename: Dhanpat K
authors.initials: DK
authors.lastname: Kochar
authors.forename: Ashis
authors.initials: A
authors.lastname: Das
mesh_terms: D060085:Coinfection / Q000175:diagnosis / Q000469:parasitology; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D020459:Genes, rRNA; D006801:Humans; D007194:India; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D010963:Plasmodium falciparum / Q000145:classification* / Q000235:genetics; D010966:Plasmodium vivax / Q000145:classification* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012339:RNA, Ribosomal, 28S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D012339:RNA, Ribosomal, 28S